Enzyme Activity Lab 13 AP Biology (Peroxidase + Hydrogen Peroxide → Complex → Peroxidase + Water + Oxygen) 2H2O2 → 2H2O + O2 (gas)

Learning Objectives • The student is able to design a plan for collecting data to show that all biological systems are affected by complex biotic and abiotic interactions (2D1 & SP 4.2, SP 7.2). • The student is able to use models to predict and justify that changes in the subcomponents of a biological polymer affect the functionality of the molecule (4A1 & SP 6.1, SP 6.4). • The student is able to analyze data to identify how molecular interactions affect structure and function (4B1 & SP 5.1). ■

Basic background information • Basic protein structure • The concept of induced fit • The role of enzymes • That structure, function, and environment are all required for maximal function of enzymatic reactions

2H2O2 → 2H2O + O2 (gas) Peroxidase is an enzyme that breaks down peroxides, such as hydrogen peroxide, and is produced by most cells in their peroxisomes. Peroxide is a toxic byproduct of aerobic metabolism. Various factors — abiotic and biotic — could have a major influence on the efficiency of this reaction.

Prepare Read your lab hand out and prepare for the lab by making notes about the procedure in your lab notebook. Remember to number the pages and always date the pages when entries are made. Think about how you will get things done in the short amount of time we have!!!

safety Safety for this lab: You will wear goggles, aprons and tie long hair back. Remove lose jewelry. Make sure clothing is not going to get in the way. We will be using acids and bases, guaiacol, which is poisonous if swallowed and it is flammable. Know the location of the eye wash, fire blanket and safety shower.

Never mix syringes or mouth pipette Never mix syringes or mouth pipette. Pour small quantities of the substances into cups or beakers that are labeled to pipette or use to extract from using the syringe. Clean all counter tops with Clorox wipes following the lab. Wash your hands following the lab.

Label carefully Gather all materials Plan your procedure and know what you are doing before you start. Do a mock run… There is timing involved, so be prepared to start the timer as soon as you mix the materials. Make data charts ahead of time so you have a place to put your results.

So, what is this lab about? Turnip peroxidase is the enzyme that catalyzes the reaction that breaks down hydrogen peroxide into water and oxygen.

guaiacol We can “see” the reaction because we are adding guaiacol, an indicator of oxygen due to a color change that occurs in its presence. The more oxygen the deeper the brown the color becomes. The compound guaiacol has a high affinity for oxygen, and in solution,it binds instantly with oxygen to form tetraguaiacol, which is brownish in color. The greater the amount of oxygen produced, the darker brown the solution will become.

We can qualitatively or quantitatively measure the color change after allowing the reaction to occur. A color palette is prepared by placing different amounts of enzyme and substrate mixture with distilled water so that the final percent of the solutions varies by 10% in each of the 11 test tubes prepared. This will provide a way to view the different colors that can be seen for the different amounts of oxygen released at maximum production. It will be used for comparison for the other reactions.

Baseline is a universal term for most chemical reactions Baseline is a universal term for most chemical reactions. In this investigation, the term is used to establish a standard for a reaction. Thus, when manipulating components of a reaction (in this case, substrate or enzyme) you have a reference point to help understand what occurred in the reaction. The baseline may vary with different scenarios pertinent to the design of the experiment, such as altering the environment in which the reaction occurs. In this scenario, different conditions can be compared, and the effects of changing an environmental variable (e.g., pH) can be determined.

Color palette

Rate can have more than one applicable definition because this lab has two major options of approach, i.e., using a color palette and/or a spectrophotometer to measure percent of light absorbance. When using a color palette to compare the change in a reaction, you can infer increase, decrease, or no change in the rate; this inference is usually called the relative rate of the reaction. When using a spectrophotometer (or other measuring devices) to measure the actual percent change in light absorbance, the rate is usually referred to as absolute rate of the reaction. In this case, a specific amount of time can be measured, such as 0.083 absorbance/minute.

The tubes can be placed in a spectrophotometer and the absorbance measured at a wavelength of 436 nm or they can be used qualitatively to match colors with the tubes from the next part of the lab. The tubes contain a 10% increase of enzyme substrate mixture as they increase: Tube 0 0% oxygen, Tube 1 10%, 2 20%, etc.

Part 1 Baseline materials+ labeling 1. E= enzyme the cold, turnip enzyme label a 2.5 ml syringe to use to measure this. 2. P= product (oxygen) which is shown when guaiacol reacts with it and turns brown label a 2.5ml syringe to use to measure this 3. NB= buffer pH7 neutral buffer label a 10ml syringe to use to measure this 4. S= substrate, Hydrogen peroxide 5. test tube- label SPNB substrate, product, neutral buffer 6. Test tube- label ENB enzyme, neutral buffer

1 2 3 4 5 B ENB A SPNB 3ml NB neutral Buffer pH 7 Time minutes 1 2 3 4 5 Scale/ number B ENB 3ml NB neutral Buffer pH 7 1ml E= Enzyme turnip peroxidase A SPNB 1ml NB neutral Buffer pH 7 1ml P = guaiacol 2ml Substrate hydrogen peroxide Cover with parafilm and mix . Use a disposable pipette to transfer tube A to tube B. Cover and mix. Immediately observe by comparing to the color palette and begin timing! Observe every minute for 5 minutes. Calculate the rate for the baseline. Color change/% oxygen over time

1 2 3 4 5 B ENB A SPNB 3ml NB neutral Buffer pH 7 Time minutes 1 2 3 4 5 Scale/ number B ENB 3ml NB neutral Buffer pH 7 1ml E Enzyme turnip peroxidase A SPNB 1ml NB neutral Buffer pH 7 1ml P = guaiacol 2ml Substrate hydrogen peroxide Immediately observe by comparing to the color palette and begin timing! Observe every minute for 5 minutes. Calculate the rate for the baseline. Color change/% oxygen over time

Part 2 This part will test how different pH levels will alter the reaction rates as compared to the baseline. Gather your materials. Plan your procedure. Put in the correct amounts. Be careful with the buffers! Acids and bases can harm your skin and eyes! Label the test tubes,Set up your test tubes in pairs.

Tubes 1,2,4,9,11,12 Make a data chart! Tubes 3,5,6,7,8,10 1 ml NB neutral buffer 1 ml P=product indicator guaiacol 2 ml S= hydrogen peroxide Make a data chart! Tubes 3,5,6,7,8,10 1 ml E=turnip peroxidase solution 3 ml buffer of the correct pH for the tube. For example tube 3 use pH 3, tube 5 pH of 5 etc!

Observe at time zero and every minute for 5 minutes! Tubes 1,2,4,9,11,12 1 ml NB neutral buffer 1 ml P=product indicator guaiacol 2 ml S= hydrogen peroxide Be ready! Mix tube 1 with tube 3. Observe at time zero and every minute for 5 minutes! Tubes 3,5,6,7,8,10 1 ml E=turnip peroxidase solution 3 ml buffer of the correct pH for the tube. For example tube 3 use pH 3, tube 5 pH of 5 etc!

rate Calculate the rate for each tube. How much oxygen produced in 5 minutes? Based on the readings over the 5 minute time period compared to the color change or % of oxygen. % change over time= rate

graph Rate for each pH

Assessment Questions Complete the assessment questions in your lab book. #3. If you omitted the enzyme? If you omitted the substrate? If you omitted the indicator? Based on your answer to #4 develop a specific question to test for part 3 of the lab. Your group must submit an experimental plan for approval.

Part 3 Write your procedure in your lab book. Write your data in your lab book. Data analysis What conclusion can be drawn from your groups data?

conclusion What did you learn from this lab overall? What errors occurred, variable were uncontrolled? How can these be improved on next time? Remember to sign and date your lab pages. Add the lab to your table of contents and number the pages. Entries should be made in ink. You may add graphs by taping them into the lab book.

If a spectrophotometer is available, the following information is useful. The use of measuring devices can better quantify your results. Using a spectrophotometer, you can select a specific wavelength to fit the color/pigment expected in an experiment. The change in the amount or concentration of color/pigment may be measured as absorbance (amount of the wavelength trapped by the pigment) or transmittance (amount of the wavelength that is not trapped by the pigment). For Procedure 1: 1. Turn on your spectrophotometer approximately 10 to 15 minutes prior to starting the investigation so that it will warm up appropriately. 2. To measure the amount of the compound tetraguaiacol, set the wavelength to 470 nm. Or 436? Lab book

3. Set your machine at zero absorbance using a blank containing all the appropriate materials except the substrate (i.e., 13.3 mL of distilled water, 0.2 mL of guaiacol, and 1.5 mL of enzyme extract = 15 mL total).